Computational Ecology and Software, 2016, 6(2): 55-65

Article

Fluctuating asymmetry in evaluating the developmental instability of

Glossogobius giuris (Hamilton, 1822) from Lake Mainit, Surigao del
Norte, Philippines

C.C.D.Joseph1, J.H. Jumawan1, B.J. Hernando1, L.Z. Boyles2, J.C. Jumawan1, J.P.B.Velasco1,
C.C.Cabuga1, S.O.M.A. Abastillas1, E.A. Requieron2, M.A.J. Torres3
1
Biology Department, College of Arts and Sciences, Caraga State University – Main Campus, Ampayon, Butuan City 8600,
Philippines
2
Science Department, College of Natural Sciences and Mathematics, Mindanao States University – General Santos City Campus,
9500 Fatima, General Santos City, Philippines
3
Department of Biological Sciences, College of Science and Mathematics, Mindanao State University - Iligan Institute of
Technology, Iligan City, Philippines
E-mail: candyclaire13@yahoo.com

Received 23 February 2016; Accepted 23 March 2016; Published online 1 June 2016

Abstract
This study was conducted to assess the status of Lake Mainit by using fluctuating asymmetry in the
populations of Glosogobius giuris. A total of 200 G. giuris was collected with 100 per sex. All the samples
were placed in a flat styrofoam for the pinning of its fins to make it wider and to clearly see the samples point
of origin for the land-marking process. 10% Formalin was applied in all the fins of the fish samples to make it
hardened using a small brush. Twenty (20) landmarks were used to analyze the body shape of the fish. Several
studies proved that FA can be used to directly assess water quality and the overall status of the ecosystem.
Using thin-plate spline (TPS) series, landmark analyses were obtained and subjected to Symmetry and
Asymmetry in Geometric Data (SAGE) software. Results in Procrustes ANOVA showed that individual
symmetry showed a highly significant difference (P = 0.00) as well as Sides (Directional Asymmetry) and
Interaction (Fluctuating Asymmetry) in the study area. All the samples showed FA in both sexes. The results
of Principal Component Scores displayed a higher percentage in female (69.2797%) than male (63.9214%)
from Lake Mainit. Variations are almost found in all the body part of the goby in female and male gobies
except for anterior insertion of second dorsal fin and superior margin of the preoperculum. Females have a
higher fluctuating asymmetry than in males. Females spawn and males protect the eggs from any predators. In
this case, females are more susceptible to stress because they have to forage in order to compensate the energy
used up during spawning. Males are less susceptible because they usually stay on the nesting ground, and don’t
have to cover far distances in finding food. This study validates the use of FA in determining the status of the
fishes caught connecting to what the status of the environment is based on the result.

Keywords environmental condition; bio indicators; morphological variations.

Computational Ecology and Software 
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Editor­in­Chief: WenJun Zhang 
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56 Computational Ecology and Software, 2016, 6(2): 55-65

1 Introduction
The environment has been frequently disturbed due to anthropogenic activities caused by humans. Some of
these factors include climate change which subjects the environment to extreme temperatures and there is also
pollution. Many studies have concluded that pollution eventually weakens or destroys the normal ecosystem
which supports human health, production of food and biodiversity (Tare and Bhojwani, 2012).
Fluctuating asymmetry refers to small abnormalities from a prior expectation of symmetric development
in morphological traits (Swaddle, 2003) which will be measuring the difference between traits on the left- and
right-hand side of a bilaterally symmetrical organism (Moller and Swaddle, 1997). It is a quantitative
biomarker for it detect individual and population stress produced by physicochemical contaminants during the
developmental process (Pankakoski et al., 1992) as well as natural disturbances that can lower the number of
population of distinct fish (Badyaev et al., 2000). In the case of a polluted ecosystem, fishes were observed to
have high fluctuating asymmetry that lives in stressful environments considering they have to compensate
stress by requiring energy (Natividad et al., 2015).
Fish is one of widely used bio indicators known, they are valuable bio indicators because they are
relatively easy to determine in their numbers, biological diversity and behaviors. Also, they are less sensitive
than lower organisms to natural micro-environmental changes, which makes them suitable for the evaluation
of macro-environmental changes (Gadzała-Kopciuch et al., 2004). Fish growth is considered as a biomarker
for riverine pollution because it integrates all effects within the fish (Shakir and Qazi, 2013). Declining of the
production of fish and dominance of physical deformities are the major signs of deteriorating genetic make-up
(Natividad et al., 2015). Glossogobius giuris or the Tank goby is the main focus of this study as a bioindicator
in the two areas. Since G. giurisis good sensitive indicator (Venkataraman et al., 2007), any morphological
variation of its body will indicate a stress (Natividad et al., 2015). Tank goby is common and abundant in Lake
Mainit (Tumandam et al., 2005).
Lake Mainit is the fourth largest lake in the Philippines which is geographically located in the Province of
Surigao del Norte and Agusan del Norte on the Island of Mindanao. According to Mosende and Mozol (2011),
Lake Mainit has been identified to have a rich biodiversity, particularly on aquatic resources. Threats and
disturbances are also present in the lake. The mouth of the lake outlet is becoming shallow which prevents
rapid exit of the water causing it to flood in the lower portions of the lakeshore. Another disturbance is the
erosion from denuded mountainside where timber and mining companies operate. Mining waste, domestic
sewage, fertilizer, and pesticides are also serious pollutants in the lake. Gobies found in lake Mainit had a size
range of 57-242 mm, which is generally smaller now than in 1990’s (de Guzman, 2008)
This paper aims to determine the morphological variations in the body shape between sexes of
Glossogobius giuris found in Lake Mainit using fluctuating asymmetry. According to Unito-Ceniza (2012), it
is important to study the morphological variations in the body shapes of fishes using fluctuating asymmetry to
provide a better understanding of the genetic structure of fish species, it also be an awareness of the status of
the environment, and provide scientific basis for formulating a comprehensive management plan for fisheries.

2 Materials and Methods

2.1 Study area
This study was conducted in Lake Mainit which lies between 9°27’07.45” N 125°30’39.79” in Surigao del
Norte, Philippines. Fish collection was done using nets with small holes in the month of September, 2015.

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Fig. 1 Map showing Lake Mainit, Surigao del Norte, Philippines.

2.2 Fluctuating asymmetry of Tank Goby in Lake Mainit

2.2.1 Sample processing
Fish samples that used in this study were purchased from the local fisher folk in the area, prior to its species
with the total of 200 individuals (100 male & 100 female). Samples were placed in styro box with ice water to
maintain its freshness. The collected samples will then be process for image capturing and analysis.
The sample fish was placed in a flat styrofoam for the pinning of its fins to make it wider and to clearly
see the samples point of origin for the land-marking process. 10% Formalin was applied in all the fins of the
fish samples to make it hardened using a small brush. Digital image of the left and right lateral side of each
sample was taken using Samsung camera (14 mega pixels).
Sex of the specimens were also determined. The sex was identified after careful examination of the
specimen’s genitalia. Females were easily identified by the presence of its eggs and ovaries, which is in
general yellow or orange and granular in appearance. The males on the other hand, were determined based on
the presence of their testes which were typically smooth, whitish and non-granular in appearance eggs
(Requieron et al., 2010).Images were assorted according to sex and digitized using the tpsDig2 program
(version 2.0, Rohlf, 2004) and were saved as TPS file.

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2.2.2 Landmark selection and digitization

These images were tri-replicated for the purpose of determine digitizing error prior to asymmetry analysis.
These images were then converted to TPS format using tpsUtil program (Fig. 2) and subjected to tpsDig2
program (version 2.0, Rohlf 2004) for the assignment of landmarks. The coordinates that were used to analyze
the body shapes of the fishes using tpsdig2w32 were extracted from a total of twenty landmarks, the locations
of which are shown in Table 1 and Fig. 3.

Table 1 Description of the landmark points according to Dorado et al. (2012).

COORDINATES LOCATIONS
1 Snout tip
2 Posterior end of nuchal spine
3&4 Posterior & anterior insertion of 1st dorsal fin
5&6 Posterior & anterior insertion of 2nd dorsal fin
7&9 Dorsal and ventral insertion of caudal fin
8 Lateral line
10 & 11 Posterior & anterior insertion of anal fin
12 Insertion of the pelvic fin
13 Insertion of the operculum at the lateral profile
14 Posterior extremity of premaxillar
15 Anterior margin through midline of orbit
16 Posterior margin through midline of orbit
17 Superior margin of the preoperculum
18 Point of maximum extension of operculum on the lateral profile
19 & 20 Superior and inferior insertion of the pectoral fin

2.2.3 Shape analysis

The coordinates data taken from both sides of the bodies of the fishes was then subjected to Symmetry and
Asymmetry in Geometric Data (SAGE) (version 1.04, Marquez, 2007) software to get the principal
components which implies the deformation grid of the individual asymmetry (Natividad et al., 2015) and also
the data of procrustes ANOVA was also be obtain for further data analysis. Procrustes ANOVA tests are
performed to assess the significance of symmetry (= individual), directional asymmetry, and fluctuating
asymmetry of shape and size, given samples with three replicates per specimens. Both object (bilaterally
symmetric structures) and matching (bilaterally symmetric parts) types of symmetry are handled. Covariance
matrix correlations are also computed between symmetric and asymmetric components of variation. SAGE
allows saving symmetrized datasets.
2.2.4 Intraspecific variation between sexes
The comparisons between male and female sexes and individual symmetry were examined using the
Paleontological Statistics (PAST) software (Hammer et al., 2001). Significant statistical representations such as
box plots, histograms, and scattered plots were generated.

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Fig. 2 (A)Landmark points of G. giuris samples and (B) overview of the schematic flow of shape analysis using SAGE
Software.

3 Results and Discussion

Table 2shows the result obtained from Lake Mainit. The Procrustes ANOVA suggested an evidence for FA in
one of the factors considered. Three factors were analyzed for FA and these were individuals, sides, and
interaction of individuals and sides. The individual symmetry among the fish samples showed highly
significant difference (P<0.0001). It was also observed to have a highly significant difference (P<0.0001)
between sides and interaction of individuals and sides. The results implied that asymmetry is found in
individuals and in sides (Left and right). All factors were observed to be asymmetric showing morphological
changes that might be caused by high exposure of pollutants (Natividad et al., 2015). Reducing the survival
rate over the population (Schwindt et al., 2014), and might affect the fishes’ reproduction and their
development (Bonada and Williams, 2002). The summary of Procustes ANOVA was shown in Table 2.

Table 2 Procrustes ANOVA for shape of G. giuris in terms of sexes in Lake Mainit.
EFFECT SS dF MS F P-VALUE
Female
Individuals 0.5639 3564 0.0002 3.2735 0.0001**
Sides 0.0463 36 0.0013 26.6256 0.0001**
Individual x Sides 0.1722 3564 0 9.8775 0.0001**
Measurement Error 0.0705 14400 0 -- --
Male
Individuals 0.5954 3564 0.0002 3.3451 0.0001**
Sides 0.0518 36 0.0014 28.8028 0.0001**
Individual x Sides 0.178 3564 0 14.0884 0.0001**
Measurement Error 0.051 14400 0 -- --
**highly significant (P<0.0001)

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A total of 69.2797%of FA interaction from upper 5% effective principal components (PC1-PC4) of G.

giurisin female is measured. Table 3 shows the Principal components for the variation for male and female
samples. According to the results in PC1, asymmetry can found greatest in the area covered by landmark: 1
(snout tip), 2 (posterior end of nuchal spine), 3 & 4 (posterior and anterior insertion of 1st dorsal fin), 5
(posterior insertion of 2nd dorsal fin), 7 & 9 (dorsal and anterior insertion of caudal fin), 8 (lateral line), 10 &
11 (posterior and anterior insertion of anal fin), 12 (insertion of pelvic fin), 13 (insertion of the operculum at
the lateral profile), 14 (posterior extremity of premaxillar), 15 & 16 (posterior & anterior margin through
midline of orbit), 18 (point of maximum extension of operculum on the lateral profile), 19&20 (superior and
anterior insertion of pectoral fin). The only unaffected are 6 (anterior insertion of 2nd dorsal fin) & 17 (superior
margin of preoperculum).
FA is also seen among males with the total of 63.9214% FA interaction which is which is lower than
female, almost of the region are affected except 2 (posterior end of nuchal spine), 6 (anterior insertion of 2nd
dorsal fin) and 17 which is the superior margin of the preoperculum which are not affected by any stress in the
environment.
High level of FA in fish sample morphology could be a result of their adaptation to their stressful
environment in order to survive. Since G. giurisis good sensitive indicator (Venkataraman et al., 2007), any
morphological variation of its body will indicate a stress (Natividad et al., 2015).

Table 3 Principal component scores showing the values of symmetry and asymmetry scores with the summary of the affected
landmarks.
Individual Sides Interaction Affected
PCA (Symmetry) (Directional (Fluctuating Landmarks
Asymmetry) Asymmetry)
Female
PC1 52.7498% 100% 25.4076% 1,2,3,4,5,7,8,9,10,11,12,1
3,14,15,16,18,19,20
PC2 10.1919% 18.4708% 1,4,5,6,7,8,9,10,11,17,18,
19,20
PC3 8.008% 14.2151% 1,2,3,4,5,7,8,9,12,14,15,1
6,20
PC4 5.967% 11.1862% 1,5
76.9167% 69.2797%
Male
PC1 50.9479% 100% 20.4974% 1,3,4,5,7,8,9,11,12,13,14,
15,16,18,19,20
PC2 10.4175% 17.6469% 1,4,5,6,7,8,9,10,11,17,18,
19,20
PC3 9.4775% 13.6001% 1,2,3,4,5,6,7,8,9,11,12,14,
15,16,17,18,19,20
PC4 5.0209% 12.177% 1,2,3,4,7,8,9,11,12,18,19
75.8638% 63.9214%

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Females have a higher fluctuating asymmetry than in males. Study shows that morphological variations
on body shapes of fishes do adapt to its habit and habitat. Since Lake Mainit is an enclosed ecosystem, fish are
isolated geographically that tend to have morphological variations to adapt by changing necessary
morphometric. Female and male goby have different habits conforming to their positive adaptation (Unito-
Ceniza, 2012). Females spawn and males protect the eggs from any predators. In this case, females are more
susceptible to stress because they have to forage in order to compensate the energy used up during spawning.
Males are less susceptible because they usually stay on the nesting ground, and don’t have to cover far
distances in finding food.
As observed, dorsal fins, caudal fin, anal fin, pelvic fin (pectoral fin), and parts of the head are affected.
It might be affected because fins are used for swimming or locomotion, propelling, stability and protection
from predators (Crenshaw, 2009). The mobility of the fish is considered as a factor in determining direct effect
of stressor of the fish especially in the dorsal, caudal, anal and pelvic fin or pectoral fin (Natividad et al., 2015).

Fig. 3 Principal components (PC) inferred deformation grid and histogram of symmetric individuals in G. giuris(Female)
displaying distribution of asymmetry. Percentages on the sides indicate quantities of variation for which each PC account.

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Fiig. 4 Principal components

c (PC
C) implied defoormation grid an
nd histogram off individual (symmetric) in G. giuris
m species.
male

To visualize
v the actual affected landmarkss, Fig. 3 and 4 shows the actual digitizzed image of the male andd
female fishes
fi with thhe PCA deforrmation grid which displaayed the asym mmetrical shhape of G. giiuris species..
Blue marrks representeed differencees indicating fluctuation
f on
n the affectedd landmarks ssuggesting thee evidence off
FA. The identified lanndmark pointts affected byy FA were sho own togetherr with the actuual photograp
ph of the fishh
sample (Fig.
( 5). The defined illusstration was summarized
s using
u affecteed PC1 and P PC2 landmark ks because itt
was the two
t principal components to have the highest
h accouunted variationn.

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P
PC1

P
PC2

Fig. 5 Actualiized picture of digitized femalle and male fishh with the affectted landmarks shown
s in PCA- deformation grrid for PC1 andd
PC2

4 Conclu usion
Fluctuatiing Asymmettry was used in this studyy to assess Laake Mainit, whitew goby orr Glossogobiu us giuris wass
used as the sample medium
m becaause of its abundance
a in
n the said lakke of. A totaal of 200 speecimens wass
collectedd, 100 of femaale G. giuris and 100 of male.
m The resuults indicatedd evidence off FA among samples
s of G.
giuris froom Lake Maiinit of both sex,
s Procrustees ANOVA showed
s a highhly significannt differencess in the threee
factors (P<0.0001)
( which were individuals, sides, and d interaction of individuuals and sidees. Principall
componeents analysis was employyed in determ mining the affected
a landmmarks. Femaale has a hig gher affectedd
landmarkks with 69.27797%while malem has 63.99214%. Affeccted landmarrks due to FA A were seen almost of itss
entire boody. Since Laake Mainit iss an enclosedd ecosystem, introduced pollution can’t be drained outside. Thee
study dem monstrated thhe use of FA as a tool in innvestigating the
t morpholoogical asymm metry using metric
m traits off
G. giuriss samples from Lake Mainnit. The resullts of the stud dy will be immportant to the local goverrnment in thee
managem ment of the saaid Lake.

Acknow wledgement
The researchers woulld like to thannk the leaderrs B/Capt. Ro
omanito C. Amante
A Jr. andd Mr. AnolLiigtas ofBrgy..
Pagatpatan, Butuan City
C and the Biology
B Depaartment, Caraaga State Unniversity for tthe technical assistance inn
fish disseection.

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